1. Technical Field
The present invention relates to the culture, handling, preservation, storing, reconstitution, and shipping of cells and tissue. Specifically, the present invention relates to apparatus and methods for culturing and preserving cells and tissue in an enclosure without the need to remove them from the enclosure at any point during the sterilization, seeding, culturing, cryopreservation, shipping, or restoration process. Also disclosed is an apparatus and method of pipette interface with a container in a manner that blocks contaminants from entering the container.
2. Discussion of the Related Art
Tissue constructs hold promise to provide societal health care benefits. A wide variety of potentially beneficial applications are emerging, some with the potential to repair defects or abnormal tissues in the body, including those related to wound care, cardiovascular disease, and orthopedic care. These applications are anticipated to result in a tremendous amount of tissue constructs being produced annually to meet the needs of society. The tissue culture device used for tissue construct culture plays a critical role in the cost of production, which in turn impacts overall health care costs to society.
The petri dish has commonly been used to culture tissue constructs for research and production applications because of its simplicity. Unfortunately, the petri dish does not provide optimal culture conditions or allow efficient production. The petri dish is subject to contamination, automated handling is difficult, tissue constructs residing in a petri dish are prone to gradient exposure, tissue constructs must be physically handled during the manufacturing process, controlling the final shape of the tissue construct is difficult, and the petri dish is not suited to efficient process control and quality control.
Another problem of the petri dish is related to inoculation of cell attachment matrices that facilitate three-dimensional culture such as collagen. Unless the tissue construct is to be the same size and shape as the petri dish, cells generally must be placed directly onto the cell attachment matrix, and not deposited gravitationally as is the case with most cell culture devices, in order to prevent cells from coming to reside on the petri dish itself. Cells residing on surfaces other than the cell attachment matrix can negatively impact the culture. Thus, the inoculation procedure does not have tight process control since cells must be directed, usually manually, to specific locations on the cell attachment matrix. This problem is magnified as production is scaled up to produce more and more tissue constructs.
An example of a typical tissue construct culture process in a petri dish, the culture of skin, illustrates the problems. One method of culturing living skin in described by Eisenburg in U.S. Pat. No. 5,282,859. A cross-linked collagen sponge is created in a manner that renders it porous on one side and having a non-porous skin on the other side. Fibroblast cells are inoculated by “injecting” them onto the porous side of the collagen sponge, which resides in a petri dish. Culture medium is placed in the petri dish, which is incubated for 10 days while the fibroblasts proliferate. The culture medium is replaced every second day, requiring the device to be exposed to potential contamination on 5 separate occasions. It may be helpful to condition the medium by exposing it to keratinocytes for a 2-day period before use. Subsequently, the collagen sponge is turned over and the non-porous side is inoculated with keratinocytes by dispensing drops of inoculum onto various portions of the sponge. The sponge is then incubated with culture medium supplemented with fetal bovine serum for 10 days, again requiring the lid of the petri dish to be removed every other day for feeding.
In this example, the petri dish is subject to contamination due to the repeated handling needed for feeding. The delivery of cells also creates contamination risk. There is no way of controlling the amount of medium that resides on each side of the sponge because that is dictated by the density of the sponge relative to the density of the medium. When the sponge is denser that the medium, it sinks to the bottom of the petri dish. When the sponge is facing fibroblast side up, fibroblasts are exposed to the majority of medium in the petri dish. After keratinocyte inoculation, fibroblasts face the bottom of the petri dish exposing cells located towards the center of the sponge to different concentrations of medium substrates than those located towards the perimeter of the sponge, since the medium will form gradients towards the center of the sponge due to the metabolic activity of the cells. The proliferation of the cells is a function of how evenly distributed the cells are on the sponge. Therefore, outgrowth varies between each living skin construct produced in proportion to the variance in the inoculation process. If humans are depositing the cells by way of pipetting or syringe deliver, skin constructs will exhibit a high degree of variance in the initial distribution of cells, even when only one operator inoculates multiple sponges. More variance can be expected with multiple operators. Robotic dispensation reduces the variance, but increases complexity and does not diminish the exposure to contamination.
The petri dish is very poorly suited to protecting the collagen sponge or helping it retain its desired shape. The collagen sponge must be physically contacted to lift it and turn it over when the opposite side of the sponge is inoculated with keratinocytes. This risks damage to the sponge and fibroblasts and again exposes the sponge to contamination. When the culture proliferates, the sponge can contract, as collagen is known to do when cells grow upon and in it. Thus, the shape of the sponge can change and there is no control over the final shape, a particularly undesirable characteristic when creating skin constructs that may be laid side by side on a patient. This leads to an additional handling process to cut the sponge into a desired shape with all the risks of sponge damage and contamination present.
Long-term storage of the living skin cannot be done in the petri dish because the materials are not compatible with freezing. Therefore, the lid of the petri dish must again be removed and the sponge, now in a weakened condition after having been exposed to culture medium for 20 days, must be physically picked up and placed in a cryopreservation bag. Subsequently, the bag must be sealed and filled with cryopreservatives, the process again risking damage to the cells and sponge, and risking contamination. This also makes it difficult to perform quality control in a manner that is inexpensive. Even if non-destructive process control limits were met during the culture process, such as glucose and oxygen consumption targets, and those process control evaluations are not capable of detecting problems that occur once the culture is complete and the sponge is transferred to a cryopreservation bag. If damage or contamination occurs at that point, it will be expensive to detect because the skin will have to be quarantined or a high amount of destructive testing will be needed to verify the transfer procedures used for any given batch of skin were acceptable. Another potential problem in process control occurs because the amount of cryoprotectant on each side of the sponge is a function of where the sponge comes to reside in the cryopreservation bag, over which the operator has little control.
Reconstituting the skin after cryopreservation can be done by removing the skin from the cryoprotectant bag, placing it in a petri dish, and adding the appropriate medium to reconstitute it, thereby causing handling and contamination exposure. Subsequently, the sponge needs to be removed from the petri dish for use, or if not reconstituted at the site of use (i.e. the hospital), packaged in another bag for shipping. This example demonstrates that it is very difficult to establish tight process control for making tissue construct products using the petri dish and a new apparatus and method is needed.
Ideally, the apparatus and method would allow protocols that are established in the research stages to be relevant in the production stage. The simplicity of the petri dish is an advantage for those performing research scale cultures. Because the petri dish is compatible with typical equipment such as pipettes and incubators, and does not require perfusion, those options should remain available as the process is scaled up. In that manner, data generated at the research level would remain relevant as scale up occurs, and would not become irrelevant if the process were changed completely. Thus, once in the production scale, the device should be capable of operating with continuous perfusion or batch feeding. Gradient formation in the culture medium should be minimized in both batch fed and perfusion modes of operation. The improved device should have an inoculation process that is repeatable, such as the gravitational method commonly used to seed tissue culture flasks. Cells should come to reside upon the cell attachment matrix as opposed to other surfaces of the device. It should be also possible to control the final shape of the tissue construct in a manner that does not expose the construct to damage or contamination. Also, it is critical that the alternative does not repeatedly expose the tissue to contamination during the inoculation, feeding, cryopreservation, reconstitution, storage, or shipping stages. Since the possibility of exposing certain types of cells to medium containing conditioning agents may produce better tissue, as in the skin culture example of medium is conditioned by exposure to keratinocytes, the alternative device should contemplate attributes that may cost reduce this process. For example, the use of a membrane to place desired compounds and molecules in proximity of specific areas of the tissue may reduce cost by limiting the amount of those compounds and molecules needed in the device.
Attempts to address the limitations of the petri dish have been undertaken, but each attempt only addresses a portion of the problems and even when combined they do not lead to an alternative that has most of the desired attributes.
Bell, U.S. Pat. No. 4,435,102, describes a container housing a tissue for the purpose of assessing the interaction of the tissue and at least one agent. Although not directed towards overcoming the problems of tissue culture in petri dishes, the art is useful as it provides a method of controlling the amount of fluid residing above and below the tissue. This is advantageous relative to the inability of the petri dish to maintain predetermined volumes of culture medium above and below the construct. Additionally, Bell teaches a method for controlling the shrinkage of the tissue by either constraining the perimeter or allowing it to attach to a membrane for constraint. A method of constraining the controlling the shape of the tissue construct by constructing the collagen matrix in a frame of stainless steel mesh is also disclosed by Bell in U.S. Pat. No. 4,485,096.
Bell provides concepts that can be used to address some of the problems of tissue culture in petri dish. However, by applying them to the skin culture process described above, it can be seen that many problems remain. Bell does not make it clear how to minimize contamination potential throughout the inoculation, feeding, cryopreservation, reconstitution, and preservation stages. Proper oxygenation of the tissue, when cells occupy all sides of the cell attachment matrix, can only be achieved by perfusing the device with oxygenated medium. This is too complex and costly for most research environments. Unless the lower compartment is perfused with oxygen-saturated medium, cells in long-term culture will quickly deplete medium in the lower compartment of oxygen. Because of the low solubility of oxygen in medium relative to the solubility of needed substrates in medium like glucose, perfusion of the lower compartment to provide oxygen requires a much higher flow rate than if perfusion just provided substrates. That increases system complexity and cost and subjects cells to a higher rate of shear than may be desirable. If perfusion to bring oxygen is not provided once the oxygen in the lower compartment is depleted, the lower portion of the tissue can only obtain oxygen from the medium in the upper compartment. Since the cells on the upper portion of the tissue have first access to oxygen in the medium, the cells on the lower portion of the tissue are subject to oxygen concentrations that are always reduced relative to the cells on the upper portion of the tissue. Thus, without high flow perfusion, the device is no better than the petri dish for oxygenating cells at the bottom of the tissue.
In applications where the tissue is to be applied to a patient, such as living skin, Bell does not provide for a way of preparing the tissue without risking damage or increasing contamination risk. If the device were to be opened at a hospital for example, the tissue would have to be cut out of the frame constraining it. Therefore, since techniques of cutting the tissue are likely to vary from hospital to hospital, little process control is available. A controlled process would remove the tissue in the same manner each time and lead to superior and consistent overall tissue quality.
Kemp et al., U.S. Pat. No 5,536,656 describes controlling shrinkage by way of casting a collagen lattice on an acellular, hydrated collagen gel in contact with a permeable member. For some applications, this minimizes the need to constrain the collagen about the perimeter. The use of an absorbent member in the second, lower compartment in order to provide a consistent and level physical support for the collagen matrix is disclosed. Advantages are described whereby the absorbent member may create diffusional barriers to help retain desirable cell conditioning factors in proximity of the tissue. However, that same characteristic can limit transport of desired molecules and compounds to the tissue from the surrounding medium. Both the permeable membrane and the absorbent member can act to prevent inoculation of two sides of a cell attachment matrix because those members block cells from reaching the cell attachment matrix. Importantly, this is not a closed system and the risk of contamination is not diminished relative to the petri dish, and may actually be increased as two open compartments need to be manipulated. Furthermore, oxygenation of the culture is limited to diffusion of oxygen from the upper liquid/gas interface. The device does not lend itself to process control during scale up since it is not possible to measure the medium for indicators such as oxygen and glucose without taking individual samples from each device.
Peterson et al., U.S. Pat. No. 6,121,042, discloses an apparatus and method for seeding and culturing three-dimensional tissue constructs and creating a dynamic environment, placing mammalian cells under simulated in vivo conditions resulting in tissue that is more likely to display the biochemical, physical, and structural properties of native tissues than tissue cultured in a petri dish. The apparatus and methods utilize a variety of methods for physically moving the tissue. Magnetic axial loading and mechanical axial loading of the tissue by way of a piston, bellows, and flexible diaphragm, and pressure cycling the environment are described. The system is overly complex for tissue that is functionally adequate without being physically placed in tension. Thus, at the research scale, the complexity and cost are prohibitive and unnecessary for many applications. Even if the tissue loading elements are eliminated from the treatment chamber, the system is still too complex for research applications as it relies on pumps and other perfusion support mechanisms. It does not make it clear how to inoculate the tissue support matrix in a manner that achieves repeatable, uniform seeding of the type needed for applications like the production of living skin. It also does not allow removal of the construct from its constraints without risking contamination of the treatment chamber and does not indicate how to prevent damage to the construct during the removal process.
The focus is on ligaments, in which an improvement relative to the petri dish is attained due to the capability of physically stressing the ligament by altering it dimensionally. In this manner the ligament is cultured under conditions more representative of those found in vivo. However, whether or not the apparatus and method are applied to ligaments or some other tissue such as skin, many limitations of the petri dish remain. There is no ability to vary the cross-sectional area of fluid normal to the plane of the construct, remove trapped gas in a non-perfused system, alter the diffusional distance for gaseous communication with the tissue during culture, adequately oxygenate and feed the culture in the non-perfused state, direct cells to the appropriate location during seeding, make use of centrifugal force as a method of seeding a cell attachment matrix, control the final shape of tissue construct while retaining a closed system, allowing control of predetermined molecules and compounds present in proximity of the tissue. Furthermore, the apparatus and method does not contemplate the need to protect the bioreactor housing from damage during cryopreservation if the housing is comprised of a gas permeable material, capable of providing passive or non-passive gas transfer to and from the culture, but not entirely compatible with cryopreservation conditions. Also, the use of standard laboratory pipettes for liquid handling in a manner that minimizes contamination is not contemplated. The apparatus and methods are also complex and eliminate the most desirable attribute of the petri dish, which is its simplicity.